We have previously demonstrated that DHA metabolism to N-docosahexaenoylethanolamine (synaptamide) is a significant endogenous mechanism for promoting neurogenesis, neuritogenesis and synaptogensis in a cAMP dependent manner. We further demonstrated that orphan G-protein coupled receptor 110 (GPR110, ADGRF1) is the synaptamide target receptor, triggering cAMP production with low nM potency. During this period, we continued our investigation on the in vivo significance of this mechanism in injury models along with biochemical and structural characterization of the newly deorphanized GPR110. We found that intravitreal administration of synaptamide immediately after optic nerve crush (ONC) enables the robust regeneration of the lesioned optic nerve, leading to partial functional recovery of vision in adult mice through GPR110 activation. We developed a stable synaptamide analogue A8 that has significantly enhanced activity for axon regeneration and functional recovery. We also examined the effective treatment time window. Synaptamide or A8 applied at 1 h after injury enabled axon regeneration and partial restoration of vision, suggesting practical therapeutic utility of these compounds in optic nerve injury. This investigation was extended to spinal cord injury using a transection injury model. Preliminary results indicated that synaptamide injection (20 mg/kg, i.p.) improves motor function tested at 4 weeks after injury and attenuated the elevation of inflammatory marker protein expression around the injury site. We have identified the expression of gpr110 in the spinal cord and dorsal root ganglion (DRG). In cultured DRG, synaptamide stimulated axon growth in a GPR110/cAMP/PKA-dependent manner, suggesting that activation of GPR110 by its endogenous or synthetic ligand may have therapeutic potential also in spinal cord injury. Our previous findings indicated that DHA-derived synaptamide is a potent suppressor of neuroinflammation in an LPS-induced model, by enhancing cAMP/PKA signaling and inhibiting NF-kB activation through GPR110 activation. During this period, we furthered the investigation with EtOH and found that ethanol exacerbates the LPS-induced inflammatory responses both in the brain and periphery. Synaptamide effectively suppressed the ethanol- and LPS-induced injury in vivo and in vitro microglia and macrophage models, suggesting syanptamide-induced GPR110/cAMP activation as a new strategy to ameliorate the ethanol-induced disturbance of the innate immune system. GPR110 belongs to adhesion GPCRs (aGPCRs), a newly emerging class of GPCR, but its structure as well as G protein coupling is not well understood. To understand the molecular basis of the GPR110 activation, we continued to investigate the GPR110 structure by probing the conformational changes of GPR110 by in-cell chemical cross-linking and quantitative mass spectrometry. With site-directed mutagenesis and computation modeling we found a specific binding of synaptamide to the GAIN domain, which in turn induces conformational changes of GPR110 in the intracellular regions and triggers G-protein activation and beta-arrestin binding. We also investigated the interaction of GPR110 with G proteins in living cells using chemical cross-linking, immunoprecipitation and quantitative mass spectrometry, and discovered that GPR110 forms a complex with Gs but not Gq. Within 3 min of stimulation by synaptamide, Gs dissociates from GPR110 and activates downstream signal transduction evidenced by cellular phosphorylation of CREB. To our knowledge, this is the first demonstration for dynamic interaction of G proteins with an adhesion GPCR during activation in living cells. During this period, we have also investigated whether the well-recognized N-acylPE (NAPE) pathway is the primary mechanism for the production of synaptamide. We observed the presence of N-docosahexaenoylphospatidylethanolamine (NDPE), the DHA analogue of N-arachidonylphospatidylethanolamine (NArPE), in the brain homogenate and Neuro 2A cells incubated with DHA. To test the involvement of NDPE, we first established a method to sensitively detect and quantify NDPE produced in cell culture by combining silica SPE and transamidation. Since NArPE is believed to be biosynthesized via transfer of the AA group in the sn-1 position of phosphatidylcholine (PC) to PE, we prepared 1-DHA-lysoPC as a preferred substrate for NDPE production and compared the cellular production of NDPE and synaptamide from this substrate in comparison to DHA. We found that both NDPE and synaptamide production is significantly higher from free DHA in comparison to 1-DHA-lysoPC, indicating that NDPE production may take a biosynthetic route different from that of NArPE. Nevertheless, NDPE production parallels synaptamide production, suggesting that synaptamide and anandamide may also use the similar biosynthetic mechanisms. We also found that regardless of the substrate used for incubation, NDPE production is preferred from plasmalogen PE compared to diacyl PE. Among plasmalogen PE molecular species, NDPE production is strongly preferred for the species containing p16:0 at sn-1 and 18:1 at sn-2. Further investigation will lead to the knowledge that may provide insight into the potential biochemical targets besides FAAH for controlling synaptamide-mediated neurotrophic and neuroprotective function.